This invention relates to novel tracers and their synthesis and use in an immunoassay for the detection of controlled drugs such as amphetamine (APM), methamphetamine (MAPM) and their derivatives, in a biological or aqueous sample. In particular, this invention provides methods for synthesizing novel tracers in which a non-controlled substance is both the starting material in tracer synthesis and the binding site on the resulting novel tracer for the antibody, thereby eliminating the necessity of using controlled substances as starting materials. In addition, the novel tracers of the present invention can be used as an analyte analog in an immunoassay, such as a continuous flow displacement immunoassay. A continuous flow displacement immunoassay works on a principle whereby immobilized antibody is first saturated with a labeled analyte analog, the labeled analyte analog is displaced by the analyte in the testing sample, and the displaced labeled analyte analog is then measured. It was unexpectedly discovered that the novel tracers of the present invention substantially improve the performance of the continuous flow displacement immunoassay as compared with conventionally designed tracers.
Amphetamine and methamphetamine are derivatives of a compound known as a phenylethylamine. Both amphetamine and methamphetamine are stimulants of the central nervous system and of the sympathetic division of the peripheral nervous system. Like other stimulants, the short-term effects of amphetamine or methamphetamine intake include increased heart rate, increased blood pressure, reduced appetite, dilation of the pupils, feelings of happiness and power, and reduced fatigue. It is because of their stimulant effects that these compounds are abused and sold illicitly.
Due to common abuse of amphetamines and methamphetamines, there is a growing need for non-invasive, rapid tests to detect the presence of these drugs in biological specimens. In the past, amphetamines in biological samples were detected by a number of techniques such as thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), and gas chromatography/mass spectrometry (GC/MS). These assays are generally time consuming and have poor assay sensitivity. See Vapatalo, H. K. S, and Senius, K. E., Comparison of Saliva and Urine Samples in Thin-Layer Chromatographic Detection of Central Nervous System Stimulants, Intl. J. Clin. Pharmacol. Res., 4: 5-8 (1984), Cook, C. E., et al., Pharmacokinetics of Methamphetamine Self-administered to Human Subjects by Smoking s-(+)-Methamphetamine hydrochloride, Drug Metab. Dispos., 21: 717-723 (1993), Suzuki, S., et al., Analysis of Methamphetamine in Hair, Nail, Sweat, and Saliva by Mass Fragmentography, J. Anal. Toxicol., 13: 176-178 (1989), which are incorporated herein by reference as if fully set forth. In addition, they are difficult in that highly trained personnel are required to perform TLC, HPLC and GC/MS assays. Immunoassays have provided preferable alternative methods to using TLC, HPLC and GC/MS assays for the detection and quantitation of amphetamines and methamphetamines. In particular, immunoassays have improved sensitivity, efficiency and are less labor intensive. A number of immunoassay techniques for detecting amphetamines and methamphetamines in urine have been developed. See Brynes, et. al. EPO #279,213 B1, Hu et. al., EPO #371, 422A2, and Helman et. al., EPO #371,253 A2 which are incorporated herein by reference as if fully set forth. Further, the continuous flow displacement immunoassay has been demonstrated as a useful and rapid method for detecting drugs of abuse in saliva and urine. See Hao Yu et al., Use of the USDT Flow Immunosensor for Quantitation of Benzolecgonine in Urine, Biosensors and Bioelectronics, 725-734 (1996); Nam, D. et al. Programme and Abstracts of TIAFT 2000 at Helsinki, 2000; Liang, G., et al., Proc. of ICADTS 2000, Jun. 22-26, 2000, which are incorporated herein by reference as if fully set forth.
A tracer as used herein is a labeled antigen or hapten used in an immunoassay to compete with the particular substance of interest (the analyte) for antigen binding sites of an antibody. The tracer can be a labeled antigen or hapten identical to the analyte to be detected, or it can be modified in such a way that it is structurally related to the analyte and has the desired cross-reactivity toward the selected antibody. In a continuous flow displacement immunoassay for example, a modification to the labeled antigen or hapten that decreases binding affinity may actually enhance the displacement reaction, and consequently the sensitivity of the system. For detection of illicit drugs, most immunoassays generally used the illicit drugs themselves, i.e. labeled amphetamines and methamphetamines, as tracers to detect the presence and or quantity of these substances in the sample. Because these drugs are illegal, a series of procedural guidelines and paperwork accompanies their utilization in the laboratory. Recent use of non-controlled substances as starting materials in amphetamine and methamphetamine tracer synthesis has been reported (Heiman, D., et al. EP 0 371 233 A2), but the final tracer synthesized still contains the amphetamine molecule itself as the binding site for the anti-amphetamine antibodies.
As shown in FIG. 1, there are different ways of synthesizing tracers. One of the common methods of tracer synthesis involves using the illicit drugs, such as amphetamines and methamphetamines, as the starting materials. (Salamamone, S. et al, EP 0 386 644 A2) These starting materials are carried through multiple synthesis steps to yield a drug-based tracer. (See FIG. 1, Method A.) Another alternative method of synthesizing tracers involves the use of non-controlled substances as starting materials to yield a drug-based tracer. (Heiman, D. F. et al., EP 0 371 233 A2) (See FIG. 1, Method B.) In both these methods, the synthesis routes involve the illicit drugs as starting materials, intermediates or final prepared tracer.
The present invention provides methods for the synthesis of a novel set of tracers produced from non-controlled substances yielding a labeled non-controlled substance as the tracer (See FIG. 1, Method C). The synthesis of these non-drug based tracers does not involve the production of illicit drugs at any point in the synthesis process. We have found that these novel tracers are ideal for use in immunoassays detecting the presence and/or quantity of amphetamines and methamphetamines in biological or aqueous samples. We have found that it is not necessary to have the specific labeled analyte itself (i.e., amphetamine) as the tracer as long as the antibody in the immunoassay system binds to the tracer with a decreased affinity from that of the analyte in the biological samples. The novel tracers of the present invention are particularly useful in the continuous flow displacement immunoassay.
This invention relates to novel compositions suitable for use in immunoassays for detecting amphetamine (APM), methamphetamine (MAPM) and their derivatives in biological or aqueous samples. The tracer synthesis methods developed in this invention eliminate the use of the illicit drugs, i.e. amphetamine and methamphetamine, as the starting materials for or products of tracer synthesis. These non-drug based tracers are particularly useful for the continuous flow displacement immunoassay. The present invention describes the processes for synthesizing the novel tracers, and the application of these tracers in fluorescence immunoassays for the detection and quantitation of phenylethylamine derivatives in biological samples.
FIG. 2 depicts the basic structures of amphetamine and methamphetamine. Amphetamine and methamphetamine are examples of the analytes that can be detected in the immunoassays of the present invention. Amphetamine has an n value of 1. The tracers of this invention have the n value of greater than 1. The preferred tracers have an n value that is 2 or more. Thus, in one embodiment of the invention, a synthetic tracer is provided that comprises a phenylalkylamine of the formula selected from the group consisting of: 
attached to a label, wherein n greater than 1. FIGS. 3 and 4 show common fluorescent labels that can be used in the tracer synthesis of this invention.
In one embodiment of the present invention, the synthetic novel tracer is the compound, labeled 1-methyl-3-phenylalkylamine, which is depicted in FIG. 5.
In another embodiment of the present invention, the synthetic novel tracer is the compound N-labeled 1-methyl-3-phenylalkylamine, which is depicted in FIG. 6.
In another embodiment of the present invention, the synthetic novel tracer is a compound labeled N-methyl-1-methyl-3-phenylalkylamine, which is depicted in FIG. 7.
In another embodiment of the present invention, the synthetic novel tracer is a compound N-labeled N-methyl-1-methyl-3-phenylalkylamine, which is depicted in FIG. 8.
In a preferred embodiment of the present invention, the synthetic novel tracer is the reaction product of succinidyl active ester of para-hemisuccinito-1-methyl-3-phenylpropylamine-N-trifluoroacetamid with Cy5EDA shown in FIGS. 9 and 10. The preferred tracer is synthesized by the method described in Example II.